The engineering of living cells and microbes is driving a new era of cancer therapy. This transformative approach allows for the genetic programming of living cells to intelligently sense and respond to environments, ultimately adding specificity and efficacy that is otherwise unattainable with molecular-based therapeutics. Due to recent microbiome studies indicating the prevalence of bacteria within the human body and specifically in tumor tissue, bacteria have generated significant interest as cancer therapies. Additionally, a multitude of empirical studies have demonstrated that administered bacteria home and selectively grow in tumors due to reduced immune surveillance of tumor cores. Given their presence and selectivity for tumors, bacteria present a unique oppor- tunity to be engineered as intelligent delivery vehicles for cancer therapy. The objective of this proposal is to engineer and optimize S. typhimurium for metastatic colorectal cancer therapy. Since animal based-testing regimes limit the rate of clinical progress, we will use a high-throughput, bacteria-spheroid platform to rapidly test therapeutic payloads and production and release strategies. We will also assess the effect of therapies on colorectal genetic backgrounds, and investigate spatio-temporal hetero- geneity in 3D spheroids with the use of engineered cell reporters. We will then test lead candidates in mouse models of primary and metastatic colorectal cancer to evaluate safety and efficacy. We will focus on colorectal cancer due to several proof-of-concept studies from our lab demonstrating efficacy in colorectal spheroids and animal models. In particular, we showed that oral delivery of bacteria can specifically colonize colorectal liver metastases, providing an attractive delivery route as a cancer therapy. Since these metastases are often con- fined to the liver, this approach can have a significant impact on tumor growth and survival. The research in this proposal will help to establish a framework to genetically engineer microbes for cancer therapy, and significantly accelerates tools that will impact the broader cancer and synthetic biology communities. If successful, future lead candidates for potential clinical trials will be identified on the basis of therapeutic efficacy and safety studies from this proposal.